Prebiotically plausible oligoribonucleotide ligation facilitated by chemoselective acetylation

Abstract

The recent synthesis of pyrimidine ribonucleoside-2',3'-cyclic phosphates under prebiotically plausible conditions has strengthened the case for the involvement of ribonucleic acid (RNA) at an early stage in the origin of life. However, a prebiotic conversion of these weakly activated monomers, and their purine counterparts, to the 3',5'-linked RNA polymers of extant biochemistry has been lacking (previous attempts led only to short oligomers with mixed linkages). Here we show that the 2'-hydroxyl group of oligoribonucleotide-3'-phosphates can be chemoselectively acetylated in water under prebiotically credible conditions, which allows rapid and efficient template-directed ligation. The 2'-O-acetyl group at the ligation junction of the product RNA strand can be removed under conditions that leave the internucleotide bonds intact. Remarkably, acetylation of mixed oligomers that possess either 2'- or 3'-terminal phosphates is selective for the 2'-hydroxyl group of the latter. This newly discovered chemistry thus suggests a prebiotic route from ribonucleoside-2',3'-cyclic phosphates to predominantly 3',5'-linked RNA via partially 2'-O-acetylated RNA.

Figure 1. Chemoselective acetylation of RNA

a, Protection of the 2′-OH group of 1-3′P facilitates rapid template-directed 3′,5′-ligation following electrophilic phosphate activation. The 3′-OH group of 1-2′P is protected to a lesser extent such that 1>P is the major product of phosphate activation and slow template-directed 2′,5′-ligation follows. b, Treatment of adenosine-3′-phosphate (A3′P, 100 mM) with sodium thioacetate 3 (100 mM) and cyanoacetylene 4 (200mM) in D₂O at neutral pD for 24 h results in selective acetylation of the 2′-OH group. Curly arrows indicate electrophilic activation/acetylation steps. X, leaving group; Y, leaving group generated by electrophilic activation of phosphate oxygen, with or without a subsequent nucleophilic displacement; Ade, N9-linked adenine; yield judged by ¹H-NMR integration.

Figure 2. Chemoselective acetylation: mixtures and alternative electrophiles

a, Treatment of A3′P (80 mM) and A2′P (20 mM) as above (Fig. 1b) results in the exclusive 2′-acetylation of the former nucleotide. b, Partial ¹H-NMR spectrum of the reaction products described in a. c, Additional electrophiles (6-8) shown to drive the acetylation of ribonucleotides with thioacetate 3. Direct acetylation with 9 is also possible, as is oxidative activation of 3 with ferricyanide 10 to afford ferrocyanide 11 and a dimeric acetylating agent 12. Curly arrows indicate electrophilic activation/acetylation steps. Ade, N9-linked adenine; %Σnucl., yields expressed as percentage of total nucleotide, otherwise yield of product expressed as a percentage of the specific starting material from which it derives (as judged by ¹H-NMR integration).

Figure 3. Chemoselective acetylation of 3′P oligoribonucleotides expedites templated ligation

a, Sequences and reaction conditions employed for acetylation (i) and subsequent templated ligation (ii). The acetylation mixture contained 80 μM primer and 50 mM NAI 9; the ligation mixture contained 4 μM primer from the acetylation reaction, 25 μM template, 30 μM ligator, 200 mM imidazole nitrate buffer (pH 6.2), 10 mM MnCl₂, and 100 mM NCI 8. Ligation conditions were based on those reported previously for the conversion of A3′P to A>P, and for the ligation of oligomers with 5′P and 2′,3′-diol termini^,. b, Denaturing PAGE analysis of a ligation reaction (+), and controls without the reaction component indicated. The gel was imaged by UV transillumination after treatment with SYBR^® Gold nucleic acid gel stain, which does not reveal the ligator strand. MALDI-TOF MS further evidenced the formation of the monoacetylated 17 nt ligation product isolated from an excised gel band: (m/z) [M+H]⁺ calcd average mass for C₁₆₂H₂₀₂O₁₂₂N₅₉P₁₆ found 5422.42; requires 5423.24. NAI, N-acetylimidazole; NCI, N-cyanoimidazole; P, 10 nt primer; T, 13 nt template; P-L, 17 nt primer-ligator ligation product.

Figure 4. Quantification of ligation products

a, Deanturing PAGE analysis of the templated ligation (+) of the fluorescently 5′-(6FAM)-labelled 10 nt primer and 7 nt ligator (concentrations as per Fig. 3a), with controls lacking either NAI 9, NCI 8, or both. Only labelled primer and products deriving therefrom are visible. Yields are presented as mean ± s.d.; n = 3 (controls) or 6 (+). b, Time-course gel analysis for the ligation of the fluorescently-labelled primer that has been acetylated in the presence of the template and ligator strands. Concentrations for the acetylation were 6 μM primer, 38 μM template, 46 μM ligator and 50 mM NAI 9. Ligation reaction concentrations were as above (Fig. 3a). Yields are averages of three reactions. c, Graph derived from the time-course analysis (b) depicting the development of ligation product with time. Error bars are ± s.d.; n = 3. Yields were obtained by fluorescence scanning; n.d. not detectable. NAI, N-acetylimidazole; NCI, N-cyanoimidazole.

Figure 5. Chemoselective acetylation favours ligation of 3′P oligomers over 2′P oligomers

a, Denaturing PAGE analysis of reactions to assess ligation selectivity. The gel was imaged by fluorescence scanning (top), before it was stained with SYBR^® Gold and imaged by UV transillumination (bottom) to reveal the 13 nt template (primers could not be detected). Unreacted 7 nt (dye-labelled) ligator is also present in the right-hand lane. The acetylation mixtures contained 40 μM of (each of) the indicated primer(s) and 50 mM NAI 9; the ligation mixture contained 4 μM (of each) primer from the acetylation reaction, 4 μM template, 4 μM 3′-(6FAM)-labelled ligator, 200 mM imidazole nitrate buffer (pH 6.2), 10 mM MnCl₂, and 100 mM NCI 8. b, Sequences of oligomers used to assess linkage selectivity are shown (top), and an illustration of the acetylation-ligation reactions conducted with each primer pair is given (bottom), depicting the preferential ligation of 3′P oligomers. c, Mean yields (± s.d.; n = 3) of ligation products determined by fluorescence scanning. n.d. not detectable; the ligation yield in this case was below the detection limit of the fluorescence scanner when gel loading was reduced to prevent detector saturation and allow accurate quantification.

Figure 6. Templated ligation of acetylated 3′P and 2′P oligomers affords 3′,5′- and 2′,5′-linkages respectively

a, b, HPLC traces of ammonolysed (deacetylated) ligation reactions, using 3′P (a) or 2′P (b) primer, template and ligator strands (sequences as per Fig. 3a). Following ligation, ammonolysis was performed as described in the text and the mixtures analysed by HPLC (top, black). Co-injection with 17 nt product standards possessing either a 3′,5′-(middle, green) or 2′,5′-(bottom, red) linkage at the ligation position revealed the selective formation of all-3′,5′-linked RNA from the 3′P primer, and RNA 2′,5′-linked (at the ligation junction only) from the 2′P primer. Smaller amounts of 17 nt standards were used for co-injections in b to allow easier comparison with the smaller ligation product peak. The split 10 nt primer peak indicated in b consists of a mixture of 2′P- and 3′P-terminated oligomers, owing to ammonolysis of the 2′,5′-linked product and the primer 2′,3′>P. See Supplementary Fig. S20 and S21 for peak assignments; yields of 44 % and 5 % were estimated from peak integrals for 3′P and 2′P ligation respectively (adjusting to account for partial hydrolysis of the 2′,5′-linkage under ammonolysis conditions).